KR102041875B1 - Pharmaceutical composition containing cannabidiol extract and TRAIL for preventing or treating conlon cancer - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating colorectal cancer containing cannabidiol extract and trail (TRAIL: tumornecrosis factor-related apoptosis-inducing ligand) extracted from hemp, more specifically, cells of colorectal cancer cell lines By using the trail in combination with the cannabidiol extract derived from the hemp that induces death, while ensuring stability against normal cells, it is possible to significantly increase the cell death sensitivity and to increase the anticancer effect.

Description

Pharmaceutical composition containing cannabidiol extract and TRAIL for preventing or treating conlon cancer}

The present invention relates to a pharmaceutical composition for preventing or treating colorectal cancer containing cannabidiol extract and trail (TRAIL: tumornecrosis factor-related apoptosis-inducing ligand) extracted from hemp.

The incidence of colorectal cancer in Korea ranks first in the world, and the growth of colorectal cancer is accelerating due to the aging society and westernized eating habits. As a result, many social costs are incurred, and there are not many drugs available to treat colorectal cancer. Therefore, active research on colorectal cancer is necessary. Colorectal cancer is currently used in combination with anticancer drugs such as oxaliplatin and irinotecan and antibody treatments such as cetuximab or bevacizumab. The risk is even worse. In light of this, new drugs need to be discovered and developed to develop better treatments for colorectal cancer.

Cannabidiol (CBD: cannabidiol) is one of the main components of cannabis (Cannabis) is a material compared with tetrahydrocannabinol (THC: Tetrahydrocannabinol). In Korea, a lot of research is not conducted because it is designated as a drug, but in foreign countries, it is not only used for medical treatment to alleviate symptoms such as pain, memory disorders, anxiety, etc. but also active research.

CBD has a structure similar to THC, but THC is known to induce excitability and have a cell killing effect in some cancers, whereas CBD is less studied and does not induce excitability than THC.

In addition, it is reported that both THC and CBD bind to cannabinoids receptors (CB1, CB2). THC binds directly to receptor, but CBD indirectly binds to receptor.

When CBD binds to the receptor, Ceramide can be made to induce autophagy, cell death through ER stress, and cell cycle arrest through ERK.

Cancer, one of the world's deadliest diseases, is expected to increase incidence due to prolonged life expectancy along with westernized eating habits. In particular, Korea's colorectal cancer is reported to be the most common in the world, and even in the case of therapeutic drugs, it is urgent to develop new therapeutic agents because they remain in the present without further progress.

Therefore, there is a growing interest in developing technologies that use CBD as a new therapeutic agent and treatment method that can induce death of colorectal cancer and increase the effectiveness of the therapeutic agent when used in combination with currently used therapeutic agents.

Korean Patent Publication No. 2013-0067248

Accordingly, the present inventors found that cannabidiol extract derived from hemp induces apoptosis of colon cancer cell lines among natural medicines used as herbal or folk remedies, and also has a trail (TRAIL: tumor necrosis factor-related apoptosis). When used in combination with -inducing ligand), it has been confirmed that it is more effective in preventing or treating colorectal cancer by increasing apoptosis sensitivity, thus completing the present invention.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating colorectal cancer containing cannabidiol extract and trail extracted from hemp.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating colorectal cancer containing cannabidiol (cannabidiol) extract and trail (TRAIL: tumornecrosis factor-related apoptosis-inducing ligand) extracted from hemp.

The cannabidiol extract extracted from the hemp can be obtained by extraction with a solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms and mixed solvents thereof.

The concentration of the cannabidiol extract extracted from the hemp may be 0.1 to 0.4 mg / ml.

Cannabidiol extract extracted from the hemp may be contained in 0.00001 to 30% by weight of the total composition.

The trail may be contained in 0.0005 to 50% by weight of the total composition.

Cannabidiol extract extracted from the hemp may be to induce apoptosis of colorectal cancer cell lines.

The trail may be to improve the apoptosis sensitivity of colorectal cancer cell line with cannabidiol extract extracted from hemp.

A pharmaceutical composition for preventing or treating colon cancer containing cannabidiol extract and trail derived from hemp according to the present invention is used by using a combination of cannabidiol extract extracted from the hemp that induces apoptosis of colon cancer cell lines. It can increase the anti-cancer effect by remarkably increasing the cell death sensitivity while securing stability against normal cells.

1 is a result showing the effect of TRAIL and CBD on cell growth for colorectal cancer cell line and colon normal cells.
2 is a result showing the effect of the combination of TRAIL and CBD on cell growth in colorectal cancer cell lines and colon normal cells.
Figure 3 is an experimental result of the degree of improvement of the sensitivity of TRAIL by CBD when combined with TRAIL and CBD for colorectal cancer cell line.
Figure 4 is an experimental result for confirming apoptosis sensitivity enhancing factor when using a combination of TRAIL and CBD.
5 is an experimental result confirming the increase of Chop induced from ER stress by CBD in colorectal cancer cell line.

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition for preventing or treating colorectal cancer containing cannabidiol extract and trail extracted from hemp.

In the present invention, "colon cancer" refers to a malignant tumor composed of cancer cells generated in the large intestine, and may be largely divided into genetic factors and diet. Even if the food itself does not contain a direct carcinogen, carcinogens may be produced when we eat and are absorbed by the body, which is ingested by enzymes produced by the intestinal bacteria during the metabolism of dietary components. This is because it can be converted to a carcinogen.

The term "prevention" in the present invention means any action that inhibits or delays the onset of the disease by administration of the composition, "treatment" means any action that improves or benefits the symptoms of the disease by administration of the composition. do.

In the present invention, the cannabidiol extract extracted from the hemp may be extracted from one or more selected from the group consisting of hemp leaves, stems and roots, in particular, cannabidiol extract extracted from the leaves of hemp It may be advantageous in terms of apoptosis inducing effect on colorectal cancer cells.

In the present invention, the cannabidiol extract extracted from the hemp may be extracted with a solvent selected from the group consisting of water, alcohol having 1 to 4 carbon atoms and mixed solvents thereof.

In addition, the alcohol may be selected from the group consisting of methanol, ethanol and butanol, and in the case of cannabidiol extract extracted with ethanol, in particular 70% ethanol as the extraction solvent of the cannabidiol extract as an extraction solvent It can be advantageous in that it can minimize toxicity and side effects.

In addition, the concentration of the cannabidiol extract may be 0.1 to 0.4 mg / ml, preferably 0.1 to 0.3 mg / ml, more preferably 0.1 to 0.2 mg / ml, the concentration of the cannabidiol extract is If it is less than the range may be a small effect on the apoptosis on the colorectal cancer cells, if the above range may increase the toxicity and side effects to the human body.

In addition, the content of the cannabidiol extract may be included as 0.00001 to 30% by weight, preferably 0.00001 to 20% by weight, more preferably 0.00001 to 15% by weight of the total pharmaceutical composition. If the content of the cannabidiol extract is less than the above range may have a slight apoptosis effect on the colorectal cancer cells, if it exceeds the above range may increase the toxicity and side effects to the human body.

In general, TRAIL is a relatively safe and promising death ligand in clinical applications, but recently, the sensitivity of apoptosis to TRAIL depends on cancer cells, and among the cancer cells expressing TRAIL receptors, many TRAIL-resistant cancer cells have been reported. In addition, if TRAIL is continuously treated with cancer cells, cancer cell lines that were initially susceptible to TRAIL may also be resistant to TRAIL.

In order to overcome this problem, it has been reported that treatment with a concentration of TRAIL, which rarely induces toxicity to cells, can be combined with other anticancer drugs such as doxorubicin and cisplatin, which can effectively induce apoptosis by increasing sensitivity to TRAIL. However, since these anticancer agents also exhibit cytotoxicity, there is a problem of causing severe damage to normal cells as well as cancer cells. For this reason, studies are needed to find a new cancer chemopreventive agent that can be used for the treatment with TRAIL, which has low cytotoxicity and TRAIL sensitizing effect, but can minimize toxicity to normal cells.

In particular, the pharmaceutical composition using a combination of cannabidiol extract and trail extracted from hemp as described above can maximize the anticancer effect while minimizing toxicity to normal cells.

In the pharmaceutical composition according to the present invention, the content of the trail may be 0.0005 to 50% by weight, preferably 0.001 to 45% by weight, more preferably 0.005 to 40% by weight, and the content of the trail is less than the above range. , Cannabidiol cell death sensitivity improvement effect is insignificant, if the above range may have a stability problem.

The preferred dosage of the pharmaceutical composition of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for more effective prophylaxis or treatment, the pharmaceutical composition of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg, and the administration may be administered once a day. It may be administered several times.

The pharmaceutical composition of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The pharmaceutical composition of the present invention may further comprise a suitable carrier, excipient or diluent commonly used in the manufacture of the pharmaceutical composition. Preferably the composition is tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, transdermal absorbents, gels, lotions, Ointment, cream, patch, cataplasm, paste, spray, skin emulsion, skin suspension, transdermal patch, drug-containing bandage and suppository, and may be oral or It may be various parenteral formulations, but is not limited thereto.

When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose (at least one compound). lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propyleneglycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like may be used.

The present invention also provides a method of treating colorectal cancer, comprising administering the pharmaceutical composition to a suspected colorectal cancer subject.

The composition, colorectal cancer and treatment are as described above.

The method of treatment specifically comprises administering the composition to a subject suspected of having colorectal cancer in a pharmaceutically effective amount. The subject means an entire mammal including a dog, cow, horse, rabbit, mouse, rat, chicken or human, but the mammal of the present invention is not limited to the above examples. The pharmaceutical composition may be administered orally, subcutaneously, intraperitoneally, pulmonary, and intranasally, and is administered by a suitable method, including intralesional administration if necessary for local immunosuppressive treatment. Non-oral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Preferred modes of administration are intravenous, subcutaneous, intradermal, intramuscular and instillation.

The preferred dosage of the pharmaceutical composition of the present invention depends on the condition and weight of the individual, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. Since the extract of the UV-treated rice leaf of the present invention shows a high growth inhibitory effect on cancer cells, by administering a pharmaceutical composition comprising the cannabidiol extract as an active ingredient to suspected colorectal cancer, the occurrence of colorectal cancer You can also treat colon cancer by blocking its progression.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples and test examples.

Example 1 Cannabidiol Extract Preparation

Bamboo leaves and stems were powdered to obtain 100 g of hemp powder. It was deposited in 1000 g of ethanol and eluted at 40 ° C. for 48 hours. Subsequently, the solids were separated by a centrifuge, and the separated supernatant was collected and filtered. This was followed by a conventional vacuum concentration method to obtain a cannabidiol extract having a concentration of 0.15 mg / ml.

Example 2: TRAIL Preparation

TRAIL was purchased from KOMA Biotech, Seoul, Korea.

In Comparative Examples 1 and 3 below, the experiment was conducted by the following method to confirm the effect of the combined use of CBD and TRAIL on cell death. The cell lines used in Experimental Examples 1 to 5 were all purchased from ATCC (Manassas, USA).

Experimental Method

1) Cell proliferation measurement by TRAIL and CBD treatment in colorectal cancer cells

end. TRAIL and CBD Processing

① Colon cancer cell lines are treated with TRIL 6μM and CBD 4μM.

② Put the MTS solution and check the cell viability by measuring the absorbance at 550nm after 4 hours reaction.

2) Increased apoptosis induced by CBD and TRAIL in colorectal cancer cells

end. Confirmed that colony formation ability is inhibited when CBD and TRAIL are used together

① Seeding colorectal cancer cell line (DLD-1). CBD (4 μM), TRAIL (10 μg / ml).

② After 14 days colony is formed and dyed.

I. Increased sensitivity to TRAIL in CBD treatment-Flow cytometry

① Seeding colorectal cancer cell line (DLD-1). 24 hours later CBD (4 μM) is treated.

② After 20 hours, harvest TRAIL (10 μg / ml) for 4 hours and harvest.

③ Stain Annexin V-FITC (Enzo Life Sciences, Inc.), PI (propidium iodide) and check it using a flow cytometry machine.

All. Protein level confirmed increased sensitivity to TRAIL in RUNX3 overexpressed cell line-Western blotting

① Seed the colorectal cancer cell line (DLD-1) and treat CBD (4μM) after 24 hours.

② Harvest 4 hours after TRAIL treatment, collect protein through lysis and perform western blotting.

③ Confirm the change of expression using markers related to cell death.

3) Increased DR5 Expression in CBD Treatment in Colorectal Cancer Cells

end. Death receptor protein expression during CBD treatment

① Seed colon cancer cell line (DLD-1) and treat CBD with 4μM.

② Harvest after 24 hours, collect protein through lysis and perform western blotting.

I. Changes in DR5 expression during CBD treatment-Immunofluorescence

① colorectal cancer cell line with CBD 4μM.

② After 24 hours, immobilize 3.7% formaldehyde, immobilize the antibody with triton x100, and attach the primary and secondary antibodies.

③ After nuclear staining through DAPI staining, mount the slide and check DR5 expression through confocal microscopy.

Comparative Example 1: Effect of TRAIL and CBD alone on Cell Growth in Colorectal Cancer Cells

1 shows the effects of TRAIL and CBD alone on cell growth in colorectal cancer cell lines and colorectal normal cells.

Referring to A and B of FIG. 1, it can be seen that TRAIL and CBD promote cell growth for HCT116, DLD-1, and HT29, which are colorectal cancer cell lines, but for cell growth of CCD-18Co, which is normal colon cells. It can be seen that there is no great influence.

Experimental Example 1: Effect of Combination of TRAIL and CBD on Cell Growth in Colorectal Cancer Cells

2 is a result showing the effect of the combination of TRAIL and CBD on cell growth in colorectal cancer cell lines and colon normal cells.

2A is a result of confirming apoptosis effect by trypan blue staining after applying TRAIL and CBD to colon cancer cell lines HCT116, DLD-1, and HT29, and increasing the sensitivity of TRAIL by combination of TRAIL and CBD. It can be seen that, but as shown in B of Figure 2 colon normal tissue (CCD18-Co) can be seen that there is no toxicity because no cell death occurs.

Experimental Example 2: Apoptosis Effect by Combination of TRAIL and CBD

Figure 3 is an experimental result of the degree of improvement of the sensitivity of TRAIL by CBD when used in combination with TRAIL and CBD for colorectal cancer cell line.

Referring to A of FIG. 3, TRAIL apoptosis by TRAIL was increased by 10% compared to control in the DLD-1 cell line, which is a colorectal cancer cell line. TRAIL apoptosis by TRAIL was increased by more than 30% when combined with CBD. Can be.

In addition, referring to B of FIG. 3, it can be seen that the apoptosis factors PARP, Caspase-3, 9, 8 were increased by TRAIL in combination with CBD treatment in the DLD-1 cell line.

In addition, referring to C of Figure 3, as a result of treating the caspase inhibitor to confirm whether this apoptosis is caspase-dependent, it can be seen that the sensitivity of TRAIL that was increased by the CBD is lost.

From this, it can be seen that the increase in TRAIL sensitivity by CBD occurs through caspase.

Experimental Example 3: Confirmation of apoptosis sensitivity enhancing factor by the combination of TRAIL and CBD

Figure 4 is an experimental result for confirming apoptosis sensitivity enhancing factor when using a combination of TRAIL and CBD.

FIG. 4A shows Western blotting analysis of expression levels of pro-apoptotic and anti-apoptotic proteins when TRAIL and CBD are used in the HCT116 cell line, which is a colorectal cancer cell line, and DR5, which is known to be important for TRAIL sensitivity in CBD treatment, is shown in FIG. It was confirmed to increase.

4B shows that when the combination of TRAIL and CBD for colon cancer cell lines HCT116 and HT29 is used as a result of Wester blotting, DR5 is also increased.

4C shows the result of confirming the expression of DR5 through fluorescence immunostaining, and it was confirmed that DR5 was increased by CBD treatment in the same manner as the expression of protein.

4D shows that FADR suppresses DR5 expression by CBD as a result of inhibiting DR5 expression by using DR5 siRNA to confirm whether the increase of DR5 by CBD affects TRAIL sensitivity. It was confirmed through.

From this, it can be seen that the increase of DR5 by CBD increases apoptosis sensitivity by TRAIL.

Experimental Example 4: Effect of ER (endoplasmic reticulum) stress on the increase of apoptosis sensitivity enhancing factor

DR5 is known to be increased by increasing CHOP. In order to see if the increase of CHOP by CBD is increased through ER stress, an experiment was conducted to identify factors related to ER stress.

5 is an experimental result confirming the increase of Chop induced from ER stress by CBD in colorectal cancer cell line.

Referring to FIG. 5A, p-PERK is increased by CBD and CHOP is increased in HCT116, a colorectal cancer cell line, and thus, CBD induces ER stress through PERK and increases DR5 by ER stress. .

In addition, referring to B of FIG. 5, as a result of confirming the expression of PERK-EIF2α-CHOP for each time period by the CBD treatment for 24h, it can be seen that CHOP remains as the phosphor of PERK increases from the previous time period.

In addition, referring to C of FIG. 5, CHOP expression was inhibited using CHOP siRNA to confirm whether the increase of CHOP by CBD affects TRAIL sensitivity, wherein the TRAIL sensitivity increased by CBD was detected in CHOP siRNA. Blocked again by Western blot.

From this, it was confirmed that the increase of DR5 by CBD increases apoptosis sensitivity by TRAIL.

Claims (7)

Cannabidiol (cannabidiol) extract extracted from hemp and Trail (TRAIL: tumornecrosis factor-related apoptosis-inducing ligand) containing a pharmaceutical composition for preventing or treating colon cancer. The method of claim 1,
Cannabidiol extract extracted from the hemp is a pharmaceutical composition for preventing or treating colon cancer, characterized in that obtained by extraction with a solvent selected from the group consisting of water, alcohol having 1 to 4 carbon atoms and mixed solvents thereof. The method of claim 1,
The concentration of the cannabidiol extract extracted from the hemp is a pharmaceutical composition for preventing or treating colorectal cancer, characterized in that 0.1 to 0.4 mg / ml. The method of claim 1,
Cannabidiol extract extracted from the hemp is a pharmaceutical composition for preventing or treating colorectal cancer, characterized in that it contains 0.00001 to 30% by weight of the total composition. The method of claim 1,
The trail is a pharmaceutical composition for preventing or treating colorectal cancer, characterized in that contained in 0.0005 to 50% by weight of the total composition. The method of claim 1,
Cannabidiol extract extracted from the hemp is a pharmaceutical composition for preventing or treating colorectal cancer, characterized in that it induces apoptosis of colorectal cancer cell line. The method of claim 1,
The trail is a cannabidiol extract extracted from hemp colon cancer prevention or treatment pharmaceutical composition, characterized in that for improving the apoptosis sensitivity of colorectal cancer cell line.

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